1. Field of the Invention
The present invention relates to a novel protein useful as a diagnostic tool for studies and researches relating to diagnostic and therapeutic applications to tumors, including uses in detecting tumor cells, estimating cancer malignancies, etc., and/or useful in other medical and physiological uses; and to a novel gene encoding said protein. More specifically, the present invention relates to a new membrane-type protein which is one of the MMPs having the activation capability of pro-matrix metalloproteinase 2 (pro-MMP-2), i.e. an activator for pro MMP-2, provided that said protein is different from the first membrane-type matrix metalloproteinase (MT-MMP-1), and to a gene coding for said protein. The present invention also encompasses a novel matrix metalloproteinase being specifically expressed in a human tumor cell surface layer (the instant novel matrix metalloproteinase is named "membrane-type matrix metalloproteinase-3 (MT-MMP-3)"); DNA containing a nucleotide sequence coding the protein; a host cell transformed or transfected with the DNA, a process for producing the matrix metalloproteinase which comprises using said host cell, a monoclonal antibody capable of specifically binding with the matrix metalloproteinase protein, and applications of said protein and antibody.
2. Description of the Related Art
An extracellular matrix may block the transfer of tumor cells in the invasion and metastasis of tumor cells that are present in a primary nest tissue. In order for tumor cells to transfer and invade into tissues, they must deviate from the primary nest and destroy peripheral extracellular matrixes. The metastasis of tumor cells progresses via destruction of basement membranes, invasion into and effusion from blood vessels, successful implantation on secondary organs, further growth, etc. The extracellular matrix that blocks tumor metastasis is composed of various complex components, including type IV collagen, proteoglycans, elastin, fibronectin, laminin, heparan sulfate, etc. A family of enzymes, generally named "Matrix Metalloproteinase" (hereinafter briefly referred to as "MMP"), with distinct substrate specificities are responsible for the degradation of the extracellular matrix.
It has been reported that MMP includes fibroblast-type collagenase (MMP-1), 72 kDa gelatinase (referred to as type IV collagenase or gelatinase A; MMP-2), 92 kDa gelatinase (referred to as type IV collagenase or gelatinase B; MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), neutrophilic collagenase (MMP-8), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11), etc. (Crit. Rev. Oral. Biol. Med., 4: 197 to 250, 1993). These MMPs form a family, and the primary structure of genes has been already reported. The reported amino-acid sequences deduced from cDNA data of these MMPs are recognized to be homologous, which are constituted of an N-terminal signal peptide basically removed during secretion and processing, followed by a propeptide domain, a Zn.sup.+ -binding catalytic domain, a proline-rich hinge domain composed of 5 to 50 amino acids, and a C-terminal hemopexin coagulation enzyme-like domain. There is no hemopexin-like domain in MMP-7. MMP-2 and MMP-9 include a gelatine-binding domain in addition to these.
Among these MMPs, it has been reported many times that type IV collagenase (MMP-2 and MMP-9) acting on, as a dominant substrate, type IV collagen that is a principal constituent for basement membranes is highly expressed in high metastatic tumor cells and there has been suggested that tumor cells are associated with tumor invasion into basement membrane invasion (Cell., 64: 327 to 336, 1991). The regulation of MMP activation is believed to be performed in steps including at least transcription level, a step for converting a proenzyme form wherein its enzymatic activity is latent into an active enzyme form, and controls by tissue inhibitor of metalloproteinase (TIMP) being a specific inhibitor against MMPs, etc. (Trends Genet., 6: 121 to 125, 1990).
All of the MMPs are secreted as inactive zymogens. In in vitro studies, activation of MMP-1 and MMP-9 is shown to be produced with serine proteinases such as plasmin, trypsin, cathepsin G. It has also been reported that activation of MMP-9 is caused by the action of active MMP-3 (J. Biol. Chem., 267: 3581 to 3584, 1992). However, since MMP-2 has no cleavage site sensitive to the above mentioned proteinase, activation of MMP-2 is believed not to be generated thereby (Curr. Opin. Cell Biol., 5: 891 to 897, 1993).
It has also been reported that these MMPs are produced by not only tumor cells but also circumferential fibroblasts and inflammatory cells which produce distinct MMPs, respectively (Breast Cancer Res. Treat., 24: 209 to 218, 1993; and Curr. Opin. Cell Biol., 5: 891 to 897, 1993).
It has previously been reported that, among them, MMP-2 is expressed in fibroblasts at a variety of sites accompanied with remodeling of tissue constructs and its activation is specifically generated in cancer tissues exemplified by lung cancer, in comparison with normal tissue and cancer tissue MMP-2s (Clin., Exp., Metastasis, 11: 183 to 189, 1993). In MMP-9, there is a low frequency that an active type is detected. In addition, there is proved in in vitro studies that active MMP-2 is localized at the apical site of tumor invasion (invadopodia) and it is suggested that the active MMP-2 has an important role on tumor invasion (Cancer Res., 53: 3159 to 3164, 1993; and Breast Cancer Res. Treat., 53: 3159 to 3164, 1994).
Under these backgrounds, attention has been focused on the activation mechanism of MMP-2. As described previously, however, activation of MMP-1 and MMP-9 is mediated by serine proteinases such as trypsin while the activation mechanism of MMP-2 is still undisclosed. In particular, an activating factor for MMP-2 remains unidentified. When HT1080 cells (MMP-2 producing cells) are treated with concanavalin A or 12-o-tetradecanoylphorbol 13-acetate (TPA), it is known that active MMP-2 appears in cultured medium, and it is believed that MMP-2 activating factors are induced in these cells (J. Natl. Cancer Inst., 85: 1758 to 1764, 1993; and Clin. Exp. Metastasis., 11: 183 to 189, 1993). Since this MMP-2 activation is induced by cellular membrane fractions and the activation is suppressed by chelating agents or TIMP, the MMP-2 activating factors have been presumed to be a membrane-type MMP (J. Biol. Chem., 268: 14033 to 14039, 1993).
The present inventors have previously cloned novel MMP genes using genetic engineering techniques, and obtained cloned genes coding for a new MMP having a typical transmembrane (TM) domain at the C-terminus thereof and being capable of activating MMP-2 (Nature, 370: 61 to 65, 1994). In fact, when this gene is expressed in cultured cells, the gene products are localized on the cell membrane without secretion. Thus, the present inventors have named such MMP as "membrane-type MMP (MT-MMP)".
Since, as described above, for MMPs, specifically MMP-2, the active form is found specifically in tumor cells, it is increasingly recognized that such should be targeted by anti-cancer or anti-metastatic drugs. Still, since MMP-2 exists relatively homeostatically as a zymogen in normal tissues, the regulation of MMP-2 activation resides in a process of activating it to active enzymes. Therefore, it is considered that the retrieval or identification of activating factors which are keys to this is extremely important in view of markers in the diagnosis of cancers and in the determination of malignancy and targets of anti-metastatic drugs against cancers.
In addition, it has been pointed out that MMP-2 may be involved in the cleavage of .beta.-amyloid protein which is associated with the crisis of Alzheimer's diseases. The .beta.-amyloid protein is a part of amyloid protein precursors, 1/4 of .beta.-amyloid protein area is included in the membrane-spanning (or transmembrane) area of the amyloid protein precursor, and the rest is outside the cells. It has been recently disclosed that several metabolic pathways of amyloid protein precursors exist, one of which is a process including a cleavage of inner sites of the .beta.-amyloid protein area with a protease called ".alpha.-secretase" and a discharge outside cells. It has been recently found that an amyloid protein-degrading activity is present in MMP-2, with the possibility that MMP-2 would function as .alpha.-secretase or an extracellular .beta.-amyloid protein-degrading enzyme (Nature, 362 : 839, 1993). The .beta.-amyloid protein is the main component of senile macula observed in the brains of patients with Alzheimer's diseases, and forms the core of senile macula by self-aggregation and deposition thereof. Since functional reduction of .beta.-amyloid protein-degrading enzymes may occur in the brain of the patient with Alzheimer's diseases, attention is focused on MMP-2. Here, the key is a process for activating MMP-2. The MT-MMP previously identified by the present inventors (newly named "MT-MMP-1" herein) is believed to be an activating factor for MMP-2, but the existence of unknown MMPs such as MT-MMP-1 can be anticipated from the fact that a variety of components exists in the extracellular matrix. The existence of activating factors for MMP-2, other than MT-MMP-1, is still undeniable.